Methotrexate for Proliferative Vitreoretinopathy

ABSTRACT

The use of methotrexate, e.g., repeated dosing or sustained-release formulations of methotrexate, for treating or reducing risk of proliferative vitreoretinopathy (PVR) or epiretinal membranes (ERM), e.g., after surgical vitrectomy to treat retinal detachment.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/500,351, filed on Jan. 30, 2017, which is the US National Stage under§371 of International Application No. PCT/US2015/042951, filed Jul. 30,2015, which claims the benefit of U.S. Patent Application Ser. No.62/030,778, filed on Jul. 30, 2014. The entire contents of the foregoingare hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to the use of methotrexate, e.g., repeated dosingor sustained-release formulations of methotrexate, for reducing risk ofproliferative vitreoretinopathy (PVR) or epiretinal membranes (ERM)after surgical vitrectomy and/or scleral buckle to treat retinaldetachment.

BACKGROUND

Retinal detachment (RD) is an important cause of sudden visual loss inthe United States, with approximately 40,000 cases occurring annually.Permanent visual loss will result if treatment is delayed.

A retinal detachment is defined as the separation of the neurosensoryretina from the retinal pigment epithelium (RPE). In the nonpathologicstate, the retinal pigment epithelium is a continuous epithelialmonolayer occluded by tight junctions, which maintain a strictseparation of the underlying choroidal capillary beds from thephotoreceptors of the sensory retina, thus forming the outerblood-retina barrier. Its functions include the nourishment ofphotoreceptors, elimination of waste products, and reabsorption ofsubretinal fluid.

The definitive treatment of retinal detachment is surgical repair.Multiple operative techniques are available to the treatingretinologist, but the principles underlying treatment of retinaldetachment remain the same: removal of fluid from the subretinal space,relief of any existing traction, and treatment and prophylaxis againstthe underlying cause for the ingression of fluid, whether it be due to aretinal break or an exudative process.

Proliferative vitreoretinopathy (PVR) is the most common cause forfailure of retinal detachment surgery, a complication which occurs in5-10% of all retinal detachment surgeries. PVR can also occurspontaneously in the absence of surgery. PVR is most likely to developfollowing repeated surgical instrumentation of the eye, followingsignificant physiologic insult to the eye such as in trauma, as well asin retinal detachments complicated by multiple tears, giant tears,vitreous hemorrhage, or in eyes with uveitis.

A milder form of PVR, called macular pucker or epiretinal membrane(ERM), complicates the post-operative course of 20-30% of RD surgeriesand half of these are so visually distorting that patients will requiresurgery. In addition, autopsy studies show that close to 75-80% ofpatients with RD surgery have histological evidence of proliferativemembranes. This may explain why many patients do not achieve perfectvision postoperatively after RD surgery, yet do not have any clinicallyobvious ERMs. In addition, ERMs can also develop spontaneously.

No treatments to date have been found to be preventive against PVR orERMs. Once PVR or ERMs develop, surgery is the only treatment.

SUMMARY

The present invention is based, at least in part, on the development ofmethods to treat and to reduce the risk of developing PVR or ERM.

Thus, in a first aspect the invention provides methods for treating orreducing the risk of proliferative vitreoretinopathy (PVR) or epiretinalmembranes (ERM) in a subject. The methods include administering aplurality, e.g., ten or more, intravitreal injections of methotrexateover a period of at least one, two, three, or more months, given no morefrequently than weekly.

In some embodiments, each injection provides a dose of 400 mcg in 0.1 mlmethotrexate.

In some embodiments, the methotrexate is administered posterior to thelimbus.

In some embodiments, the subject is undergoing an ocular surgicalprocedure that increases the subject's risk of developing ERM or PVR,e.g., a pars plana vitrectomy (PPV), Retinal Detachment (RD) surgery;ERM surgery; scleral buckle surgery; or a procedure in the other eye. Insome embodiments, the subject requires a PPV to treat a rhegmatagenousretinal detachment secondary to trauma; preexisting proliferativevitreoretinopathy (e.g., grade C or higher); or for other indicationsassociated with high risk condition for PVR development, e.g., giantretinal tears (giant retinal tears are defined as tears involving 90° ormore of the circumference of the globe), retinal breaks larger than 3disc areas, long-standing retinal detachments, or detachments associatedwith hemorrhage.

In some embodiments, a first injection is given at conclusion of thesurgical procedure; eight weekly injections are given untilpostoperative month two; and a final tenth injection is given atpostoperative month three.

In some embodiments, the methods include administering nine consecutiveweekly injections, and a tenth injection three months after the firstinjection.

In some embodiments, the methods include administering additionalinjections monthly after the final, e.g., tenth, injection.

In some embodiments, the methods include administering one, two, three,four, five, six, seven, eight, or nine additional injections, e.g.,monthly, after the tenth injection.

In another aspect, the invention provides methods for treating orreducing the risk of PVR or ERM in a subject. The methods includeintravitreally administering a sustained release formulation ofmethotrexate over at least a three-month period.

In some embodiments, the sustained release formulation is or comprises alipid-encapsulated formulation; multivesicular liposome (MVL)formulations of methotrexate (MTX); nano- or microparticles; polyioncomplex (PIC) micelles; or bioadhesive polymers. In some embodiments,the bioadhesive polymers comprise one or more of hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), polyacrylic acid(PAA), or hyaluronic acid (HA).

In a further aspect, the invention provides methods for treating orreducing the risk of PVR or ERM in a subject. The methods includeimplanting a device for sustained release of methotrexate over at leasta three-month period into the eye of the subject.

In some embodiments, the device is non-biodegradable.

In general, in the methods described herein, the subject does not havecancer, e.g., does not have an ocular cancer, e.g., does not have ocularor B cell lymphoma. In some embodiments, the subject does not haveuveitis. In some embodiments, the methods include determining that asubject has or is at risk of developing PVR or ERM, or is about toundergo a procedure with a high risk of PVR or ERM as a side effect, andselecting the subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a flowchart showing an exemplary treatment protocol using thepresent methods.

FIGS. 2A-2C are each sets of nine images of human PVR cells plated into12 wells with 30,000 cells per well showing that methotrexate inhibitedproliferation of human proliferative vitreoretinopathy (PVR) cells inculture. The cells were treated with 100 μM, 200 μM, or 400 μMMethotrexate (MTX) as indicated, and the images were taken after 72hours (2A), 1 week (2B) or 2 weeks (2C). At 72 hours (2A), thephotomicrograph showed similar epithelioid morphology and limitedconfluence across the control plates (top row), as well as the threemethotrexate concentrations (rows 2-4). At 1 week (2B), the controlplates (row 1) showed a uniform, confluent cellular sheet whereas rows2-4, which were exposed to methotrexate 400, 200, and 100 respectively,showed growth inhibition and lack of confluency, and were lessepithelioid in appearance. At 2 weeks, the control plates (row 1)continued to be a uniform, confluent cellular sheet, whereas rows 2-4,exposed to methotrexate 400, 200, and 100 respectively, continued tohave inhibited growth and lack of confluency.

DETAILED DESCRIPTION

Proliferative vitreoretinopathy (PVR) is a common occurrence afterretinal detachment surgery. PVR is a “scarring” condition that formsinside the eye after surgery, significant trauma, or even spontaneously.Its pathogenesis is the disruption of the retinal pigment epitheliumlayer, which is associated with inflammation, migration, andproliferation of cells to the (neural) retinal surface. Over the next4-12 weeks, membranes on the surface of the retina proliferate,contract, and apply traction on the retina, which results inredetachment of the retina from the RPE. Once PVR is present and theretina detaches for a second time, it is unlikely that vision will berestored.

Pathobiology of Proliferative Vitreoretinopathy (PVR) and EpiretinalMembranes (ERM)

Epiretinal membranes (ERM) are caused by an abnormal proliferation ofcells, e.g., retinal pigment epithelial (RPE) cells, glial cells,fibroblasts, and macrophages, on the surface of the retina, typically inresponse to ocular disease; the membranes tend to contract and causepuckering and thus distortion of the macula. See, e.g., Hiscott et al.,Br J Ophthalmol. 68(10):708-15 (1984); Hiscott et al., Eye 16, 393-403(2002); and Asato et al., PLoS One. 8(1): e54191 (2013).

Like ERM, PVR is an abnormal wound healing response of the vitreous andretina, a clinical syndrome where cells with proliferative capacity,driven by inflammatory mediators, multiply on the retinal surface,contract, and eventually cause recurrent retinal detachment (RD). Thepathogenesis of PVR begins with the introduction of RPE cells into thevitreous cavity. These cells may be introduced at the time of theretinal tear itself or may be introduced iatrogenically such as throughthe use of cryotherapy or retinectomy. Studies from monkey eyes with PVRhave also postulated that the introduction of Müller cells, as well aspotentially fibrocytes, occur as well. Concomitant with the introductionof RPE cells is the introduction or upregulation of growth factors,including vascular endothelial growth factor (VEGF), platelet-derivedgrowth factor (PDGF), fibronectin, transforming growth factor-beta(TGF-β), and other mediators. This process begins an autocrine loopwhere glial, RPE, and other cellular constituents proliferate andtransdifferentiate into contractile myofibrocytes. On pathology,macrophages as well as fibroblasts are commonly identified in specimensof PVR.

It has also been proposed that, at this time of RPE disruption,inflammation plays an important role in the development of PVR.Cytokines IL-6, IL-1, TNF-alpha, and IFN-gamma have been identified inhigh concentrations in the vitreous in the early, proliferative stagesof PVR, but they decrease to normal levels in the scarring phase. Thesecytokines are not present in eyes that do not develop PVR.

The use of a pharmacologic adjuvant to prevent PVR has been an elusivegoal in ophthalmology. A general pharmacologic strategy employed inprior PVR studies has been the single intravitreal, intraoperativeadministration of a variety of agents, such as duanoribicin,5-fluorouracil (5-FU), triamcinolone, low-molecular weight heparin, andnaproxen.¹⁰⁻¹⁵ Although the clearance of vitreally delivered drugs isdependent on a number of factors including the molecular weight of thedrug, the status of the blood-retina barriers, contents of the vitreouscavity, etc., it is likely that those drugs used in these prior PVRstudies were cleared from the eye within days after theiradministration. In contrast, PVR does not become a clinicallyappreciable, pathologic entity until at least 6-8 weeks after surgery.

Methotrexate

Methotrexate is a non-naturally occurring chemically also known asN-[4-[[(2,4-diamino-6-pteridinyl) methyl] methylamino]benzoyl]-L-glutamic acid. A folate analog, methotrexate is thought toact as an anti-proliferative agent by reversibly inhibitingdihydrofolate reductase, which prevents dihydrofolate from being reducedto tetrahydrofolate, which is used in the synthesis of purinenucleotides.

Mechanisms of anti-inflammatory action are less clear, althoughmechanisms that have been proposed include its ability to enhance theextracellular concentration of adenosine, suppression ofpro-inflammatory cytokines, inducement of apoptosis of activated Tcells, and suppression of intracellular adhesion by activated T cells.

In some embodiments, the methotrexate is formulated for repeatedinjection, e.g., in Balanced Salt Solution from 25 mg vials to asterile, single-use dose of 400 mcg/0.1 ml.

In some embodiments, the methotrexate is formulated for sustainedrelease. A number of sustained release formulations of methotrexate areknown in the art, including but not limited to biodegradable implantssuch as lipid-encapsulated formulations, e.g., Depo/Methotrexate, asdescribed in Bonetti et al., Cancer Chemother Pharmacol 33:303-306(1994) and Chatelut et al., J Pharm Sci. 1994 March; 83(3):429-32;multivesicular liposome (MVL) formulations of methotrexate (MTX), e.g.,as described in WO2011143484; nano- or micropartricules, e.g.,alpha-lactalbumin microparticles, e.g., as described in Vijayaragavan etal., Int J Pharm Res 3(1):39-44 (2011) or nanoparticles of conjugatedmethotrexate-human serum albumin as described in Taheri et al., JNanomaterials 2011 (dx.doi.org/10.1155/2011/768201); polyion complex(PIC) micelles; bioadhesive polymers such as hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC) and polyacrylicacid (PAA) derivatives, as well as hyaluronic acid (HA), e.g., Lacrisert(Aton Pharma), which is a soluble hydroxy propyl cellulose ocularinsert.

Alternatively or in addition, sustained release can be achieved using asustained-release device such as intravitreal implants, e.g., asdescribed in Palakurthi et al., Current Eye Research, 35(12):1105-1115(2010) or similar to the Retisert (Bausch & Lomb), Ozurdex (Allergan);or non-biodegradable implants, e.g., similar to Iluvien (Alimera) orVitrasert (Bausch & Lomb) implants; the I-vation platform (SurModicsInc.). See also Lee et al., Pharm Res. 27(10):2043-53 (2010); Haghjou etal., J Ophthalmic Vis Res. 6(4):317-329 (2011); Kim et al., Invest.Ophthalmol. Vis. Sci. 45(8):2722-2731 (2004); and Velez and Whitcup, BrJ Ophthalmol 83:1225-1229 (1999).

Subjects

The methods described herein can be used to prevent (reduce the risk of)PVR or ERM in patients, e.g., in patients requiring pars planavitrectomy (PPV), e.g., for rhegmatagenous retinal detachment secondaryto trauma; for patients requiring PPV for preexisting proliferativevitreoretinopathy grade C or higher; and/or for patients with retinaldetachments requiring PPV for other indications associated with highrisk condition for PVR development, e.g., giant retinal tears (giantretinal tears are defined as tears involving 90° or more of thecircumference of the globe), retinal breaks larger than 3 disc areas,long-standing retinal detachments, or detachments associated withhemorrhage.

Other uses of sustained methotrexate in the eye in addition to PVRinclude the following:

Prevention of Epiretinal Membranes After Retinal Detachment (RD) Surgery

Approximately 20-30% of RD cases develop clinically perceptible ERMs.Half of these are so visually distorting that patients will requiresurgery. In addition, autopsy studies show that close to 75-80% ofpatients with RD surgery have some degree of proliferative of membranes.This may explain why many patients do not achieve perfect visionpostoperatively after RD surgery, yet do not have any ERMs grosslyperceptible to the human eye.

Prevention of ERMs that Develop Spontaneously

ERMs can develop spontaneously, which then requires surgery. If asubject developed an ERM in one eye, implanting a device to prevent ERMsin the other eye could prevent development in that eye.

Prevention of Secondary ERM After ERM Surgery

For patients who develop ERMs, these can be removed but some reoccur andrequire reoperation. Leaving an implant could prevent the recurrent ERM.

The methods described herein can include identifying and/or selecting asubject who is in need of treatment to prevent the development of PVR orERM as a result of a condition listed above (e.g., selecting the subjecton the basis of the need of treatment as a result of a condition listedabove, e.g., an increased risk of developing PVR or ERM as a result of acondition listed above). In some embodiments, the subjects treated witha method described herein do not have ocular cancers, e.g., do not havelymphoma (e.g., B cell lymphoma), and/or do not have uveitis.

The presentation of PVR clinically encompasses a wide phenotype. PVR canvary from a mild cellular haze (Grade A) to thick, fibrous membranesthat cause the characteristic stiffened funnel of the detached retina(Grade D). A number of grading systems are in use, see, e.g., Ryan,Retina, 5^(th) ed (Elsevier 2013); Retina Society Terminology Committee.The classification of retinal detachment with proliferativevitreoretinopathy. Ophthalmology 1983; 90:121-5 (1983); Machemer R,Aaberg T M, Freeman H M, et al. Am J Ophthalmol 112:159-65 (1991); LeanJ, Irvine A, Stern W, et al. Classification of proliferativevitreoretinopathy used in the silicone study. The Silicone study group.Ophthalmology 1989; 96:765-771. In some embodiments the methods includeidentifying, selecting, and/or treating a subject who has a low grade(e.g., Grade A or Grade 1) PVR, or who has ERM. In some embodiments, themethods include monitoring the subject for early signs of thedevelopment of PVR or ERM, i.e., the presence of a “vitreous haze”indicating a cellular proliferation (which may eventually develop intoan organized sheet), and administering one or more doses of MTX asdescribed herein. Although early Grade A PVR vs. an early ERM may bedifficult to distinguish from one another, eventually untreated PVR willprogress; ERMs will cause a mild traction on the macula resulting inmetamorphopsia but will not cause detachment of the retina, whereasuntreated PVR will cause detachment and eventually result in a funneled,atrophic retina. The methods can also be used to treat subjects withoutpresent signs of PVR but who are at risk for PVR or ERMs.

Methods of Treating or Reducing Risk of PVR or ERM

The methods described herein include the use of methotrexate in subjectswho are at risk of developing a first or recurring PVR or ERM, e.g., asubject who is undergoing RD surgery or ERM surgery, as described above,and in subjects who have PVR or ERM or who are at risk for developingPVR or ERMs. In some embodiments, the methods described herein includethe use of methotrexate in subjects who have undergone, are undergoing,or will undergo a pars plana vitrectomy (PPV) or scleral buckle (SB). Insome embodiments, the methods include performing a PPV, RD surgery, orERM surgery. Methods for performing these surgeries are known in theart; for example, typically, PPV is performed under local or generalanesthesia using three, 23 or 20 gauge sclerotomy ports. Any presentepiretinal membranes can be dissected, e.g., using a membrane pick andforceps. Intraoperative tissue staining, perfluorocarbons, cryopexy,endolaser, scleral buckling, and lensectomy can also be performed asneeded. Standard tamponading agents can be used, e.g., silicone oil orgas.

The methods described herein include the use of an effective amount ofmethotrexate. An “effective amount” is an amount sufficient to effectbeneficial or desired results, e.g., the desired therapeutic effect(i.e., a prophylactically effective amount that reduces the risk ofdeveloping PVR or ERM). An effective amount can be administered in oneor more administrations, applications or dosages. A therapeuticallyeffective amount of methotrexate can be, e.g., 400 μg/0.1 ml perinjection, e.g., with at least ten injections, giving a cumulative doseof 4,000 μg over ten injections; in some embodiments, the methodsinclude giving more than ten injections, for a cumulative dose of morethan 4,000 μg. In some embodiments, the methods include giving fewerthan ten injections, for a cumulative does of less than 4,000 ug. Thecompositions can be administered one from one or more times per day toone or more times per week to one or more times per month; includingonce every other day. The skilled artisan will appreciate that certainfactors may influence the dosage and timing required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present.

In some embodiments, intravitreal methotrexate injections are performedaseptically after the topical application of anaesthesia and anantiseptic agent, e.g., 5% povidone iodine, to the conjunctival sac. Insome embodiments, each subject receives an intravitreal injection ofmethotrexate, e.g., 400 mcg/0.1 ml methotrexate, 3.0 to 3.5 mm posteriorto the limbus, depending on lens status, with a 30-gauge needle.

In some embodiments, the subjects receive multiple intravitrealinjections of methotrexate during their post-operative period. The firstinjection can be administered intraoperatively; subsequently, injectionscan be administered on post-operative (post-op) weeks 1, 2, 3, 4, 5, 6,7, and 8, and on post-op month 3, for a total of 10 injections. See,e.g., FIG. 1. In some embodiments, the methods include administering themethotrexate in ten doses, or ten or more doses, or less than ten doses,over a three-month period or longer, and injections would be given nomore frequently than weekly. In some embodiments, the methods includeadditional doses at weekly, biweekly, or monthly frequency thereafterfor an additional one, two, three, four, five, six, seven, eight, nine,ten, 11, or 12 months thereafter. In some embodiments the methodsinclude ten doses over three months as shown in FIG. 1, with an optionaladditional one or more doses at monthly intervals thereafter for anadditional one, three, six, or more months thereafter.

In some embodiments, the subjects receive a sustained release implant,e.g., as described above, that will release MTX over time, e.g., over aweek, two weeks, a month, two months, three months, six months, or ayear. In some embodiments, the methods include administering subsequentimplants to provide MTX administration for at least six months, oneyear, two years, or more.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Sustained Methotrexate in the Silicone Filled PostoperativeEye at High Risk for Proliferative Vitreoretinopathy

We hypothesized that the administration of multiple, intravitrealmethotrexate injections into eyes with high-risk features forpost-operative proliferative vitreoretinopathy (PVR) development willhave improved visual outcomes, higher anatomic final reattachment rates,decreased reoperation rates, and decreased occurrence of PVR at 4 monthspostoperatively.

We have performed a small pilot study in 10 patients with retinaldetachment whom had high-risk clinical factors for developing PVR.

Patients 18 years to 89 years old of both genders were eligible for thisstudy if they required pars plana vitrectomy (PPV) for rhegmatagenousretinal detachment secondary to trauma, PPV for preexistingproliferative vitreoretinopathy grade C or higher, or if they hadretinal detachments requiring PPV for other indications associated withhigh risk condition for PVR development, i.e.: giant retinal tears(Giant retinal tears are defined as tears involving 90° or more of thecircumference of the globe), retinal breaks larger than 3 disc areas,long-standing retinal detachments, detachments associated withhemorrhage.

The PPV was performed under local or general anesthesia using three, 23or 20 gauge sclerotomy ports. Any present epiretinal membranes weredissected using a membrane pick and/or forceps. Intraoperative tissuestaining, perfluorocarbons, cryopexy, endolaser, scleral buckling, andlensectomy were performed as needed. Either silicone oil or gas was usedas the tamponading agent.

Routine post-operative visits, which involve a dilated funduscopicexamination, occured on post-operative day 1, 7, month 1, month 2, andmonth 3. Patients return to the operating room after three months forsilicone oil removal and were seen in clinic 4 months after the originalsurgery.

In addition to receiving the above-described standard of care, patientsreceived multiple intravitreal methotrexate injections during theirpost-operative period. The first injection was administeredintraoperatively and subsequently was injected on post-op week1,2,3,4,5,6,7,8 and on post-op month 3, for a total of 10 injections.

Intravitreal methotrexate injections were performed aseptically afterthe topical application of anaesthesia and 5% povidone iodine to theconjunctival sac. Each patient received an intravitreal injection of 400mcg/0.1 ml methotrexate, 3.0 to 3.5 mm posterior to the limbus,depending on lens status, with a 30-gauge needle. After injection,patients were monitored for adverse events, including a full-dilatedfunduscopic examination.

Demographics and Pre-Operative Vision

Eight men and two women enrolled into the study (Table 1). The age ofpatients ranged from 18 to 63. Two patients (#4 & #9) were enrolled withtraumatic retinal detachment (total retinal detachment, 360 degree giantretinal tear, and retina incarcerated in scleral wound after open globeinjury). The remaining 8 patients had had multiple (average 2.5) priorretinal detachments secondary to proliferative membranes. One patient(MTX08M) had significant baseline retinal comorbidity with highpathologic myopia, staphyloma, atrophy, lattice and paving stone in theoperative eye. The median pre-operative visual acuity was Hand Motionsat 2 feet.

Operative Details

Operative details of each patient are provided in Table 2. Operativetime was recorded as a surrogate for surgical complexity. All patientsunderwent vitrectomy, extensive membrane peeling, relaxing retinectomy,perfluorocarbon liquid, endolaser, and silicone oil injection. The foursubjects previously surgically intervened on had buckles that were stillproviding adequate indentation of the globe and were therefore left inplace.

Visual and Anatomical Results

Despite the extremely poor visual and anatomic prognosis of all thesubjects enrolled in the study, no subjects developed PVR whilereceiving methotrexate during the three-month treatment protocol.Interestingly, one of the trauma patients (#4) experienced massive PVRtwo weeks after completion of the injection protocol (at 3½ monthspostoperatively), but weekly examination during the study had shown noevidence of proliferating cells; this is extremely unusual and may beaccounted for by the presence of methotrexate for three months and itssubsequent absence. This patient required re-operation. Two othersubjects developed reaccumulation of fluid under the retina requiringre-operation, but no membranes were appreciated.

Safety and Adverse Events

Adverse events observed are reported in Table 3. All subjectsexperienced a degree of conjunctival hyperemia, consistent with the useof silicone oil. Superficial punctate keratopathy (SPK) was observed inone asymptomatic patient at a single clinical exam. Examination one weeklater showed a normal corneal surface and no further sequelae wereobserved. Follow up duration in our 10 patients ranged from 4 months to39 months, with a median follow up time of 25 months. Even after monthsto years of follow up data, no significant adverse events were observed.Visual acuity and intraocular pressure at the last follow up visit wassimilar in all patients to the visual acuity and pressure observed atpost-operative month 4, at the conclusion of the study period,suggesting good long term safety.

In addition, in the same patient who experienced SPK, elevatedintraocular pressure was documented (44 mm Hg by Goldmann Tonometry) ata single examination. Per patient report, she had been taking Flonase(fluticasone nasal) “many times per day” and had been taking Pred Fortedrops QID, although she had been instructed to take Pred Forte BID. Herpressure was normalized in the exam room with topical therapy. Theintravitreal injection was administered as scheduled, and she wasdischarged from clinic with normal pressure. She was given aprescription for Alphagan and Xalatan with plans to follow up with theGlaucoma service. No further elevated pressures were observed during thecourse of the study or in follow up, suggesting that her transientelevated pressure was most likely due to excessive steroid use assuspected. Patient MTX08M ended the study with NLP vision. He had ahistory in the operative eye of pathological myopia, staphyloma,atrophy, lattice, and pave stoning. One month after his surgery (histhird intravitreal surgery in that eye and the study surgery date), discpallor was noted. Optical coherence tomographic pictures of that retinabefore and after had noted disorganized laminae, secondary to hisunderlying retinal disease. His vision at this was noted to be LP. Hecontinued to receive injection with limited improvement in his visionand noted to be NLP at the final visit.

In addition to the excellent anatomical results seen, with virtually noPVR developing during the study protocol, the protocol was associatedwith excellent visual results. The median post-operative visual acuitywas 20/200. This is notable because although one other group has usedmethotrexate in eyes with a wide variety of conditions, including PVR,no one has previously demonstrated improved outcomes. Hardwig et al.injected methotrexate intravitreally of varying doses into 5 patients,however, only one patient had any improved vision and there was nochange in visual acuity for the group (Hardwig et al., Retina28:1082-1086 (2008)). In addition, this study did not attempt tovalidate efficacy. Also, in 2006 Hardwig et al. injected 1 dose ofmethotrexate into the anterior chamber of one patient with PVR, but notinto the intravitreal space as described herein (Hardwig et al., Am JOphthalmol 2006; 142:883-885 (2006)).

The present results are very encouraging and are unlikely explained bychance. The patients who have received 10 intravitreal methotrexateinjections have done remarkably well given the severity of theircondition, as we intentionally selected patients who were at the highestrisk for PVR.

Example 2 Sustained Methotrexate Inhibits the Growth of ProliferativeVitreoretinopathy In Vitro

We hypothesized that methotrexate would inhibit human proliferativevitreoretinopathy (PVR) cells in culture. PVR membranectomy wasperformed in patients undergoing retinal detachment repair secondary toPVR. Using cellular separation techniques, cellular constituents of thePVR membranes were separated from the extracellular matrix membranes.30,000 cells per well were placed into a standard 12 welled plate. All12 wells received endothelial cell growth medium with supplementalgrowth factors. Four arms were designated consisting of three wellseach. The first arm served as a control receiving the standard growthmedium but no other intervention. The remaining wells were designated astreatment arms. The second arm of three wells served as the firsttreatment arm and exposed the cells to 400 micrograms of methotrexate.The third and fourth arms of three wells each exposed the cultured cellsto 200 and 100 micrograms, respectively, of methotrexate.

At 72 hours, similar growth of PVR cells with typical epithelioidmorphology and limited confluence was observed (FIG. 2a ). One weekafter seeding (FIG. 2b ) the PVR control cells demonstrated a confluentcellular sheet whereas cells exposed to methotrexate at allconcentrations demonstrated growth inhibition, lack of confluency, andwere less epithelioid in appearance. At two weeks (FIG. 2C), inhibitionof cellular confluency continued whereas the control plates continued toproliferate.

TABLE 1 Patient demographics Previous number of Critieria ChildbearingOcular surgeries attempted studyid Age Male Race for entry potentialcomorbidities to repair RD Patients MTX01F 47 No White PVR Grade C Nonone 3 enrolled MTX02M 60 Yes White PVR Grade C No none 3 in MTX03M 56Yes White PVR Grade C No lattice, mild cataract 3 year 1 MTX04M 25 YesWhite RD associated No Marginal lid laceration 0 with open of rightlower lid with globe injury canilicular involvement MTX05M 29 Yes WhitePVR Grade C No subluxated lens OS, lens 2 removal age 4, diagnosedglaucoma age 11, post capsule removal age 17 subluxated lens OD, lensremoval age 17 Patients MTX06M 54 YES White PVR Grade C No none 3enrolled MTX07M 57 YES White PVR Grade C No none 2 in MTX08M 63 YesWhite PVR Grade C No High Pathologic myopia; 2 year 2 staphyloma OU;atrophy OU; lattice and paving stone OS MTX09M 53 Yes White RDassociated No none 0 with open globe injury MTX10F 18 NO White PVR GradeC Yes, 2 methods of s/p 5 prior strabismus 0 contraception + surgeriespregnancy test prior to every injection

TABLE 2 Clinical results Visual Visual Acuity, Last Total Visual IOP atAcuity, Post-op Initial Final follow months acuity at last studyidinitial month 4 IOP IOP Injections Detachments up date of follow up lastfollow up follow up Patients MTX01F HM 20/200 22 13 10 0 Jul. 9, 2015 3920/150 12 enrolled MTX02M 640 20/320 12 12 10 0 May 20, 2015 38 20/50015 in year 1 MTX03M HM 20/200 9 12 10 0 Jul. 14, 2015 39 20/300 16MTX04M LP 20/160 10 9 10 0* Jul. 1, 2015 37 HM 9 MTX05M 600 20/200 11 1010 0 Dec. 3, 2012 4 20/200 9 Patients MTX06M LP 20/252 soft 8 10 0 Jul.1, 2015 27 CF 9 enrolled MTX07M HM 20/200 12 12 10 0 Apr. 8, 2015 24 CF22 in year 2 MTX08M CF NLP 14 15 10 0 Sep. 17, 2014 15 NLP 14 MTX09M LP20/65 soft 9 10 0 Apr. 29, 2015 22 20/70 16 MTX10F HM CF 15 13 9 0 Mar.24, 2015 16 CF 14 *Massive PVR membranes developed two weeks after thefinal injection of methotrexate, despite there being no PVR in thepreceding three months Injections: Total number of injections ofmethotrexate 400 mcg/0.1 ml Detachments: Number of detachments due toPVR during study period

TABLE 3 Adverse events observed in subjects Superficial ElevatedConjunctival Punctate intraocular Reoperations Date of studyid hyperemiaKeratopathy pressure NLP required reoperation Indication for reoperationPatients MTX01F 7 1 1 0 0 — — enrolled MTX02M 7 0 0 0 0 — — in year 1MTX03M 5 0 0 0 0 — — MTX04M 4 0 0 0 1 Post-op month 4 Outside theinjection period (post-op month 3.5), significant PVR was seen. Patientwas taken to the OR to peel membranes and remove traction from theretina. MTX05M 7 0 0 0 1 Post-op month 2.5 Persisitent sub-retinalfluid, no membranes Patients MTX06M 5 0 0 0 0 — — enrolled MTX07M 4 0 00 0 — — in year 2 MTX08M 6 0 0 1 0 — — MTX09M 6 0 0 0 0 — — MTX10F 5 0 00 0 — —

REFERENCES

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Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating or reducing the risk ofproliferative vitreoretinopathy (PVR) or epiretinal membranes (ERM) in asubject, the method comprising administering a plurality of intravitrealinjections of methotrexate over a period of at least one, two, three, ormore months, given no more frequently than weekly.
 2. The method ofclaim 1, comprising administering ten or more intravitreal injections ofmethotrexate.
 3. The method of claim 1, wherein each injection providesa dose of 400 mcg in 0.1 ml methotrexate.
 4. The method of claim 1,wherein the methotrexate is administered posterior to the limbus.
 5. Themethod of claim 1, wherein the subject is undergoing an ocular surgicalprocedure that increases the subject's risk of developing ERM or PVR. 6.The method of claim 5, wherein the ocular surgical procedure is a parsplana vitrectomy (PPV), Retinal Detachment (RD) surgery; ERM surgery;scleral buckle surgery; or a procedure in the other eye.
 7. The methodof claim 6, wherein the subject requires a PPV to treat a rhegmatagenousretinal detachment secondary to trauma; preexisting proliferativevitreoretinopathy; or for other indications associated with high riskcondition for PVR development.
 8. The method of claim 7, wherein theindication associated with high risk condition for PVR development is agiant retinal tear, a retinal break larger than 3 disc areas, along-standing retinal detachment, or a detachment associated withhemorrhage.
 9. The method of claim 5, wherein: a first injection isgiven at conclusion of the surgical procedure; eight weekly injectionsare given until postoperative month two; and a final tenth injection isgiven at postoperative month three.
 10. The method of claim 1,comprising administering nine consecutive weekly injections, and a tenthinjection three months after the first injection.
 11. The method ofclaim 1, comprising administering additional injections monthly afterthe tenth injection.
 12. The method of claim 11, comprisingadministering one, two, three, four, five, six, seven, eight, or nineadditional injections after the tenth injection.
 13. The method of claim12, wherein the additional injections are administered monthly.
 14. Amethod of treating or reducing the risk of proliferativevitreoretinopathy (PVR) or epiretinal membranes (ERM) in a subject, themethod comprising intravitreally administering a sustained releaseformulation of methotrexate, or implanting into the eye of the subject adevice that provides sustained release of methotrexate over at least athree-month period.
 15. The method of claim 14, wherein the sustainedrelease formulation is or comprises a lipid-encapsulated formulation;multivesicular liposome (MVL) formulations of methotrexate (MTX); nano-or microparticles; polyion complex (PIC) micelles; or bioadhesivepolymers.
 16. The method of claim 14, wherein the bioadhesive polymerscomprise one or more of hydroxypropyl methylcellulose (HPMC),carboxymethylcellulose (CMC), polyacrylic acid (PAA), or hyaluronic acid(HA).
 17. The method of claim 14, comprising implanting into the eye ofthe subject a device that provides sustained release of methotrexateover at least a three-month period.
 18. The method of claim 17, whereinthe device is non-biodegradable.
 19. An implanted ocular device forsustained release of methotrexate for treating or reducing the risk ofPVR or ERM in a subject, wherein the device releases methotrexate overat least a three-month period into the eye of the subject.
 20. Thedevice of claim 19, wherein the device is non-biodegradable.